Composition for delivery of hematopoietic growth factor

ABSTRACT

A hematopoietic growth factor delivery composition includes a hematopoietic growth factor, a liquid vehicle, a first biocompatible polymer and a second biocompatible polymer. The composition exhibits reverse-thermal viscosity behavior, due to interaction between the first biocompatible polymer and the liquid vehicle. The second biocompatible polymer helps to protect the first biocompatible polymer from being dissolved in vivo following administration to a host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/073,153 entitled “COMPOSITION FOR DELIVERY OF HEMATOPOIETIC GROWTHFACTOR” filed Mar. 5, 2003; which is a continuation of U.S. patentapplication Ser. No. 09/893,339 entitled “COMPOSITION FOR DELIVERY OFHEMATOPOIETIC GROWTH FACTOR” filed Jun. 26, 2001; which claims apriority benefit under 35 U.S.C. §119(e) to U.S. Provisional PatentApplication No. 60/214,298 entitled “COMPOSITION AND METHOD FOR DELIVERYOF HEMATOPOIETIC GROWTH FACTOR” filed Jun. 26, 2000 and to U.S.Provisional Patent Application No. 60/274,891 entitled “COMPOSITION ANDMETHOD FOR DELIVERY OF HEMATOPOIETIC GROWTH FACTOR” filed Mar. 9, 2001.The entire contents of all and every part of each of the foregoingreferenced applications is incorporated herein by reference as if setforth herein in full.

FIELD OF THE INVENTION

The present invention relates to compositions for delivery ofhematopoietic growth factors.

BACKGROUND OF THE INVENTION

Functionally, hematopoietic growth factors can be considered to belongto one of three groups. The first or multilineage group includesinterleukin 3 (IL-3) and granulocyte macrophage colony stimulatingfactor (GM-CSF) which act on early colony forming units (CFU's)including colony forming unit-granulocyte, erythrocyte, megakaryocyte,macrophage (CFU-GEMM), colony forming unit-granulocyte-macrophage(CFU-GM), burst forming units erythrocyte (BFU-E) or megakaryocytes,(BFU-MK). The second or unilineage group includes erythropoietin (EPO),granulocyte colony stimulating factor (G-CSF), interleukin 5 (IL-5),macrophage colony stimulating factor (M-CSF) and thrombopoietin (TPO),and act on later hematopoietic progenitors (i.e., colony forming uniterythrocyte (CFU-E), colony forming unit megakaryocyte (CFU-Mk), andcolony forming unit eosinophil (CFU-Eo). The third or “potentiating”group includes interleukin 6 (IL-6), interleukin 11 (IL-11), lymphocyteinhibitory factor (LIF), fibroblast growth factor basic (FGFb), stemcell factor (SCF) and Flt3 ligand (Flt3-L), and act to potentiate theactivities of other hematopoietic factors. Within the third group, SCFand Flt3-L both show marked activity on hematopoietic stem cells andthus have been considered special circumstance/stem cell growth factors.

G-CSF and GM-CSF are two commonly used hematopoietic growth factors. Theprincipal action of G-CSF is the stimulation of colony forming unitgranulocyte (CFU-G), which in vivo manifests into an augmentedproduction of polymorphonuclear leukocyte (neutrophil) as well asenhancing the phagocytic and cytotoxic functions of neutrophils ingeneral. G-CSF has been shown to be effective in the treatment of severeneutropenia following autologous bone marrow transplantation andhigh-dose chemotherapy. GM-CSF and G-CSF are each used to decrease theperiod of neutropenia seen during this type of therapy and therebyreduces morbidity secondary to bacterial and fungal infections. Whenused as a part of an intensive chemotherapy regimen, G-CSF can decreasethe frequency of both hospitalization for febrile neutropenia andinterruptions in life-saving chemotherapy protocols. G-CSF also hasproven to be effective in the treatment of severe congenitalneutropenias. In patients with cyclic neutropenia, G-CSF therapy, whilenot eliminating the neutropenic cycle, will increase the level ofneutrophils and shorten the length of the cycle sufficiently to preventrecurrent infections. G-CSF therapy can improve neutrophil counts insome patients with myelodysplasia or marrow damage. The neutropenia ofAIDS patients receiving AZT also can be partially or completelyreversed.

G-CSF is typically administered by subcutaneous injection or intravenousinfusion at a dose of 1 to 20 μg/kg per day. The distribution andclearance rate from plasma (half-life of 3.5 hours) are similar for bothroutes of administration. A continuous, 24-hour intravenous infusion canbe used to produce a steady-state serum concentration of the growthfactor. As with GM-CSF therapy, G-CSF is given daily following bonemarrow transplantation or intensive chemotherapy will increasegranulocyte production and shorten the period of severe neutropenia. Inbone marrow transplantation and intensive chemotherapy patients,continuous daily administration for 14 to 21 days or longer may benecessary to correct the neutropenia. With less intensive chemotherapy,fewer than 7 days of treatment may be needed.

Both G-CSF and GM-CSF will increase the number of marrow progenitorcells in the circulation, a particularly valuable function in patientspreparing for stem cell collection. Post-transplant infusions ofharvested stem cells together with G-CSF or GM-CSF may reduce theseverity of the post-transplant neutropenia.

One hematopaetic growth factor that has recently received considerableattention for its unique properties is Flt3-L. Flt3-L is a transmembraneglycoprotein of approximately 30 kDa. Mouse and human Flt3-L sharesignificant homology at the amino acid level (70%), and showcross-species reactivity, so testing human Flt3-L in mouse produces thesame or similar biological effects as would occur in the human. Cellsknown to express Flt3-L include human and mouse T cell lines, as well asarchitectural cells of the bone marrow, specifically the bone marrowfibroblast.

Some of the myelopoietic, or white blood cell potentiating effectsattributed to Flt3-L include: 1) an expansion of CD34+CD38− cell numberwhen used in conjunction with SCF and IL-3; 2) an increase in highproliferative potential colony forming cells (HPP-CFC) and CFU-GMnumbers; and 3) in the presence of GM-CSF, the formation of largenumbers of CFU-GM. Individual and direct myelopoietic effects of Flt3-Linclude an increase in CFU-GM, CFU-GEMM and HPP-CFC survival and apreferential induction of macrophages under certain conditions. Flt3-Lalone apparently has minimal or no effects on erythroid andmegakaryocyte progenitors.

There is substantial data showing that the system of Flt3-L and itsreceptor also plays an important role in lymphopoiesis, the processesinvolved in normal growth and maturation of lymphocytes. This importantactivity has been confirmed in mice made deficient for Flt3-L System. Inthese mice hematopoietic populations are essentially normal but markeddeficiencies of early B cell progenitors are found in the bone marrow.This has led to the suggestion that Flt3-L, perhaps expressedconstitutively by bone marrow fibroblasts, is a normal regulator of Bcell lymphopoiesis, while cytokines produced by activated lymphocytessynergize with Flt3-L in times of stress to accelerate B celldevelopment

In addition to its effects on hematopoietic cells and B cells, Flt3-Lhas also been shown to stimulate the production of dendritic cells, ahighly specialized cell involved in antigen presentation and therefore,normal immunity. Also, with the observation that Flt3-L stimulates theproduction of dendritic cells, Flt3-L has been identified for potentialuse in the area of vaccines, both traditional delivery of heat killed orotherwise attenuated agents, as well as protein, peptide or DNAvaccines.

For additional information on Flt3-L, see, for example, Shurin et al.,“FLT3: Receptor and Ligand. Biology and Potential Clinical Application”,Cytokine & Growth Factor Reviews, Vol. 9, No. 1, pp. 37-48, 1998.

One of the problems associated with the hematopoietic growth factorssuch as G-CSF, GM-CSF, SCF and Flt3-L, is the need for multiple dailyinjections. This, in turn leads to another common disadvantage ofcurrent injectable therapies such as these, that being the creation of asaw-toothlike effect of plasma drug levels. This is due to the creationof large bolus bursts of drug shortly after injection, leading tosupraphysiologic levels of drug, followed by rapid drops in plasma druglevels as the drug is cleared from the body by normal clearanceprocesses. Upon the next injection, the pattern is repeated with largespikes in plasma levels followed by sub-therapeutic levels until thenext injection. An additional problem with current hematopoietic growthfactor therapy includes fever and mild-to-moderate bone pain in patientsreceiving high doses over a long period. In addition, local skinreactions and mild to moderate splenomegaly have been reported.

There is a significant need for improved formulations and methods fordelivery of hematopoietic growth factors that address one or more ofthese problems, especially as treatments involving the use ofhematopoietic growth factors continue to expand.

SUMMARY OF THE INVENTION

The hematopoietic growth factor delivery composition of the presentinvention provides for sustained delivery of hematopoietic growthfactors, thereby advantageously increasing the plasma half-life ofhematopoietic growth factors, and thereby also reducing the number ofadministrations, and therefore the number of injections, required fortreatment. Moreover, the saw-tooth profiles of drug plasma levelsexperienced conventionally should be reduced with less frequentadministrations, as should side effects caused by the frequentinjections with conventional treatments. Furthermore, it has been foundin at least some cases, that the activity of the hematopoietic growthfactor is significantly improved when administered in the composition ofthe present invention, relative to conventional formulation andadministration. Therefore, not only should fewer administrations berequired for a treatment program, but less hematopoietic growth factorshould also be required in many instances, which would be expected togenerally reduce the severity of side effects.

The hematopoietic growth factor delivery composition of the presentinvention comprises a hematopoietic growth factor, a first biocompatiblepolymer, a second biocompatible polymer and a liquid vehicle. The firstbiocompatible polymer and the liquid vehicle interact in such a mannerand are present in such proportions that the composition exhibitsreverse-thermal viscosity behavior, in that the viscosity of thecomposition increases with increasing temperature over at least sometemperature range. The second biocompatible polymer is a, protectivecolloid.

The reverse-thermal viscosity behavior of the delivery compositionpermits the delivery composition to be administered to a host as alower-viscosity flowable medium, which then converts to ahigher-viscosity form in vivo. The hematopoietic growth factor is thenadvantageously released in a sustained manner from the protectiveenvironment of the higher-viscosity form of the delivery composition. Toaccomplish this result, the delivery composition should exhibitreverse-thermal viscosity behavior over at least some temperature rangebelow the physiologic temperature of the host. The presence of thesecond biocompatible polymer helps to protect the composition frompremature degradation in vivo due to invasion by aqueous biologicalfluids, such as are encountered by the delivery composition inside thehost after administration. The inclusion of the second biocompatiblepolymer, therefore, is important to help protect the deliverycomposition so that the delivery composition can successfully make thetransition from the lower-viscosity flowable medium to thehigher-viscosity form following administration. Also, the secondbiocompatible polymer helps to inhibit premature dissolution in vivo ofthe higher-viscosity form, thereby promoting a prolonged release of thehematopoietic growth factor. Surprisingly, the inclusion of the secondbiocompatible polymer has also resulted in an observed significantincrease in the activity of the hematopoietic growth factor under atleast some circumstances. Although the mechanism of this enhancement isnot well understood, the enhancement in activity of the observedhematopoietic growth factor with the composition is significant andsurprising.

In one embodiment, the composition exhibits a reverse-thermal gelationproperty, which is a special case of reverse-thermal viscosity behaviorin which the higher-viscosity form of the delivery composition is a gel(i.e., gelatinous substance). In this preferred embodiment of thedelivery composition, the composition should have a reverse-thermalliquid-gel transition temperature that is no higher than the physiologictemperature of the host. The composition is then administerable to thehost as a flowable medium at a chilled temperature, and as the deliverycomposition warms in the host following administration the deliverycomposition converts to the gel form. Because the gel form is typicallysubstantially immobile, the hematopoietic growth factor is releasedwithin the host at the desired location from the protective environmentof the gel to facilitate sustained delivery of the hematopoietic growthfactor.

In one preferred embodiment, the delivery composition is in the form ofa flowable medium at least at a first temperature and in the gel form atleast at a second temperature that is higher than the first temperature,but not higher than the physiologic temperature of the host. Forexample, when the delivery composition is intended for use by a humanhost, the first temperature could advantageously be below 20° C.,preferably in a range of 10° C. to 20° C., and the second temperaturecould advantageously be in a range of 25° C. to 37° C. In any event,with a human host the delivery composition should be preferably in thegel form at 37° C. Also, at the first temperature, the firstbiocompatible polymer is preferably substantially entirely dissolved inthe liquid vehicle in the form of a solution that is liquid and flowableto an extent to impart sufficient fluidity to the delivery compositionso that the delivery composition is administrable to a host byinjection. The hematopoietic growth factor may also be dissolved in thesolvent, or may be in the form of a fine precipitate suspended by thehematopoietic growth factor/solvent solution. The second biocompatiblepolymer will typically be in the form of a “colloidal solution” in theliquid vehicle, at least at the first temperature.

Also, for enhanced performance, the hematopoietic growth factor shouldbe uniformly dispersed throughout the gel, which can typically beaccomplished by mixing the composition at a temperature at which thefirst biocompatible polymer/liquid vehicle combination is in the form ofa flowable liquid solution of the first biocompatible polymer in theliquid vehicle. In this way the hematopoietic growth factor can bedissolved in or uniformly dispersed throughout the solution, and thenthe temperature of the composition can be raised to convert thecomposition to the gel form for storage prior to use.

In another aspect, the invention includes a method for manufacturing acomposition in which the hematopoietic growth factor is dissolved in ordispersed throughout a solution of the first biocompatible polymer and asolvent for the first biocompatible polymer. The solvent is typically anaqueous liquid.

In yet another aspect, the present invention provides a method forpackaging and storing the hematopoietic growth factor in the protectiveenvironment of the delivery composition. Handling and storage may be ina gel or liquid form, as desired.

Both the foregoing summary description and the following detaileddescription are exemplary and are intended to provide explanation of theinvention as claimed. Other aspects and novel features will be readilyapparent to those skilled in the art from the following detaileddescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot in relation to Example 2 showing pharmacokineticprofiles of G-CSF formulated in buffer (Buffer Formulation) andaccording to one embodiment of the invention (Invention Formulation)described in Table 1. G-CSF in each of the formulations was administeredto Balb/c mice as a single intramuscular (i.m.) injection containing 7μg G-CSF. The concentration of G-CSF in serum was measured via ELISA.Reported are averages of serum G-CSF levels in 4 mice per sampling time.

FIG. 2 is a timeline of the study design for Example 2 employed toassess the pharmacological action of G-CSF in mice. G-CSF in the BufferFormulation was injected twice daily for 3 days and once the day ofsampling for a total of 7 injections. G-CSF in the Invention Formulationwas administered as a single injection on day 1 of the study withsampling at day 4. Injections were given intramuscularly (i.m.) toBalb-C mice.

FIG. 3 is a bar graph in relation to Example 2 of the pharmacologicalaction of G-CSF as determined by mobilization of HPP-CFC cells inperipheral blood. G-CSF in the Buffer Formulation was injected twicedaily for 3 days and once the day of sampling for a total of 7injections. Each injection contained either 0.1, 0.5, or 5.0 μg G-CSFfor a total cumulative dose of 0.7, 3.5, or 35 μg G-CSF/mouse,respectively. G-CSF in the Invention Formulation was administered as asingle injection on day 1 of the study with sampling at day 4. Thesingle injection contained either 0.7, 3.5, or 35 μg G-CSF. Injectionswere given intramuscularly (i.m.) in Balb/c mice. Mobilization ofHPP-CFC was determined by quantitating the number of HPP-CFC cells uponcytokine (IL-3)-stimulation of isolated peripheral blood leukocytes.

FIG. 4 is a plot in relation to Example 4 of the pharmacokineticprofiles of Flt3-L parent, Flt3-L in PGZ-1, and Flt3-L in PGZ-2. Thevarious formulations (described in Table 2) were injectedintramuscularly (i.m.) into Balb/c mice and the levels of Flt3-L inserum were followed for up to 24 hours for the parent formulation orevery 24 hours up to 96 hours for the PGZ formulations.

FIG. 5A in relation to Example 4 is a bar chart showing white blood cellcounts (WBC) and FIG. 5B is a bar chart showing spleen cell counts (SPC)determined in Balb/c mice following intramuscular (i.m.) injection ofVehicle control, Flt3-L parent, Flt3-L in PGZ-1, or Flt3-L in PGZ-2.Values are Mean±SEM of cell counts determined 96 hours followingadministration of the various formulations.

DETAILED DESCRIPTION

As used herein, “CFU” means colony forming unit, “CFU-GEMM” means colonyforming unit-granulocyte, erythrocyte, megakaryocyte, macrophage,“CFU-G” means colony forming unit-granulocyte, “CFU-GM” means colonyforming unit-granulocyte-macrophage, “CFU-E” means colony formingunit-erythroid, “CFU-MK” means colony forming unit-megakaryocyte, and“CFU-Eo” means colony forming unit-eosinophil.

As use herein, “BFU” means burst forming unit, “BFU-E” means burstforming unit-erythroid, and “BFU-MK” means burst formingunit-megakaryocyte.

As used herein, “IL” means interleukin.

As used herein, “EPO” means erythropoietin.

As used herein “TPO” means thrombopoietin,

As used herein, “CSF” means colony stimulating factor, “G-CSF” meansgranulocyte colony stimulating factor, “M-CSF” means macrophage colonystimulating factor, and “GM-CSF” means granulocyte-macrophage colonystimulating factor.

As used herein, “CFC” means colony forming cells and “HPP-CFC” meanshigh proliferative potential colony forming cells.

As used herein, “FGF” means fibroblast growth factor and “FGFb” meansfibroblast growth factor basic.

As used herein, “LIF” means leukocyte inhibitory factor

The terms “reverse-thermal viscosity property” and “reverse-thermalviscosity behavior” each refer to a property of a material, such as thefirst biocompatible polymer or the delivery composition as the case maybe, to undergo a viscosity increase with increasing temperature acrossat least some temperature range.

As used herein, “reverse-thermal gelation property” refers to a propertyof a material, such as the first biocompatible polymer or the deliverycomposition, as the case may be, to change from a flowable medium,typically a liquid form, to a gel form as the temperature is raised frombelow to above a reverse-thermal liquid-gel transition temperature.

As used herein, “reverse-thermal liquid-gel transition temperaturerefers to a temperature at which, or a temperature range across which, amaterial, such as the first biocompatible polymer or the deliverycomposition, as the case may be, changes physical form from a flowablemedium, typically a liquid form, to a gel form, as the temperature ofthe material is increased.

The term “reverse-thermal gelation polymer” refers to a polymer capableof interacting with a liquid vehicle so that the polymer/liquid vehiclecombination exhibits a reverse-thermal gelation property at least atsome proportions of the polymer and the liquid vehicle.

As used herein, “biocompatible” means not having toxic or injuriouseffects on biological function in a host.

As used herein, “protective colloid” means a hydrophilic polymer thathas colloidal-size molecules and that is capable of interacting withwater molecules through hydrogen bonding. By “colloidal-size,” it ismeant that one or more of the molecular dimensions when dispersed in anaqueous liquid are in a range of one nanometer to one micrometer.

The present invention provides a composition for delivery ofhematopoietic growth factor to a biologic host, preferably a mammalianhost, and more preferably a human host. The composition comprises atleast one hematopoietic growth factor, at least one liquid vehicle, atleast a first biocompatible polymer and at least a second biocompatiblepolymer that is different than the first biocompatible polymer.Optionally, the composition may also comprise additives such aspenetration enhancers and protective stabilizers, and/or an active agentin addition to the hematopoietic growth factor.

The hematopoietic growth factor included in the delivery composition maybe any material capable of stimulating hematopoietic cell activity inthe intended host. The delivery composition may include only one type ofhematopoietic growth factor or may include more than one different typeof hematopoietic growth factors.

Exemplary hematopoietic growth factors useful in the deliverycomposition of the present invention include those in the multilineagegroup (including IL-3 and GM-CSF), the unilineage group (including EPO,G-CSF, IL-5, M-CSF and TPO) and the “potentiating” group (includingIL-6, IL-II, LWF, FGF b, SCF and Flt3-L).

The amount of hematopoietic growth factor in the composition of thepresent invention varies depending on the nature and potency of thegrowth factor. In most situations, however, the amount of hematopoieticgrowth factor in the composition will be smaller than about 20% w/wrelative to the first biocompatible polymer.

The present invention provides a hematopoietic growth factor deliverycomposition for prolonged, or sustained, delivery of hematopoieticgrowth factor, thereby reducing the frequency of administrations as partof a treatment. Furthermore, it has been found that the delivery systemof the present invention, in at least some circumstances, results inenhanced cell generation relative to the same quantity of hematopoieticgrowth factor administered by a conventional method. Not to be bound bytheory, but to aid in the understanding of the invention, it is believedthat the composition of the present invention reduces or eliminatesdegradation of the hematopoietic growth factor and allows for arelatively slow sustained administration of hematopoietic growth factorsto the host. In addition, it is believed that the composition may betargeting the hematopoietic growth factor to tissues that would make themost efficient use of the hematopoietic growth factor.

The liquid vehicle may be any suitable liquid or mixture of liquids, butis typically an aqueous liquid. An important aspect of the deliverycomposition of the present invention is that the liquid vehicle and thefirst biocompatible polymer are selected and included in the deliverycomposition in such proportions that the delivery composition exhibitsreverse-thermal viscosity behavior over at least some temperature range.Therefore, the viscosity of the delivery composition increases withincreasing temperature over some temperature range. At a firsttemperature within the temperature range, the delivery composition is ina lower-viscosity form, in which the delivery composition is in the formof a flowable medium. At a second temperature in the temperature range,which second temperature is higher than the first temperature, thedelivery composition is in a higher-viscosity form that has asignificantly higher-viscosity than the lower-viscosity form. Preferablythe viscosity of the higher-viscosity form is at least 1 times, morepreferably at least 2 times, and even more preferably at least 3 timesas great as the viscosity of the lower-viscosity form. Advantageously,the first temperature is below the physiologic temperature of the hostand the second temperature is at or below the physiologic temperature ofthe host. In this way, the delivery composition is administerable to thehost as a flowable medium in the lower-viscosity form at a chilledtemperature, with the delivery composition converting to thehigher-viscosity form as the delivery composition warms up inside thehost following administration. By “flowable,” it is meant that thedelivery composition is sufficiently fluid so as to be syringable.

The first biocompatible polymer in the delivery composition of thepresent invention typically is a reverse-thermal gelation polymer. Thefirst biocompatible polymer and the liquid vehicle are selected, and thedelivery composition is formulated with relative proportions of theliquid vehicle and the first biocompatible polymer, so that the deliverycomposition exhibits reverse-thermal viscosity behavior across at leastsome temperature range, preferably a temperature range below 40° C.,more preferably a temperature range below 37° C. and even morepreferably a temperature range within a range of from 10° C. to 37° C.Typically, the delivery composition exhibits reverse-thermal viscositybehavior over at least some temperature range within a range of 1° C. to20° C. Due to the reverse thermal viscosity behavior of the deliverycomposition, the delivery composition can be administered to the host ata cooler temperature where the composition has a lower-viscosity, withthe viscosity of the composition then increasing in the host followingadministration, whereby the mobility of the composition is severelyreduced within the host following administration. In one embodiment, inthe case of a human host, the delivery composition is preferably in theform of the lower-viscosity flowable medium at least at a firsttemperature at or below 20° C., and more preferably in a range of 1° C.to 20° C., and the delivery composition is preferably in thehigher-viscosity form at least at a second temperature in a range offrom 25° C. to 37° C.

In one preferred embodiment, the liquid vehicle and first biocompatiblepolymer are selected and included in the delivery composition in suchproportions that the delivery composition has a reverse-thermal gelationproperty, so that the higher-viscosity form is a gel. In this situation,the delivery composition should have a reverse-thermal liquid-geltransition temperature that is no higher than the physiologictemperature of the host, but that is high enough to be convenient foradministration to the host in the form of a flowable medium.

When the delivery composition has a reverse thermal gelation property,then the delivery composition will exist in the form of a flowablemedium at least at a first temperature and in the form of a gel at leastat a second temperature that is higher than the first temperature.Preferably both the first and second temperatures are below 40° C., andmore preferably the second temperature is no higher than 37° C.,especially in the case of a human host. A preferred situation is whenthe first temperature is in a range of 1° C. to 20° C. and the secondtemperature is in a range of 25° C. to 37° C.

Any first biocompatible polymer may be used that, as formulated in thedelivery composition, is capable of interacting with the liquid vehicleto impart the desired reverse-thermal viscosity behavior to the deliverycomposition. Non-limiting examples of some reverse-thermal gelationpolymers useful for preparing the delivery composition include certainpolyethers (preferably polyoxyalkylene block copolymers with morepreferred polyoxyalkylene block copolymers includingpolyoxyethylene-polyoxypropylene block copolymers referred to herein asPOE-POP block copolymers, such as Pluronic™ F68, Pluronic™ F127,Pluronic™ L121, and Pluronic™ L101, and Tetronicm T1501); certaincellulosic polymers, such as ethylhydroxyethyl cellulose; and certainpoly (ether-ester) block copolymers (such as those disclosed in U.S.Pat. No. 5,702,717). Pluronic™ and Tetronic™ are trademarks of BASFCorporation. Furthermore, more than one of these and/or otherbiocompatible polymers may be included in the delivery composition toprovide the desired characteristics and other polymers and/or otheradditives may also be included in the delivery composition to the extentthe inclusion is not inconsistent with performance requirements of thedelivery composition. Furthermore, these polymers may be mixed withother polymers or other additives, such as sugars, to vary thetransition temperature, typically in aqueous solutions, at whichreverse-thermal gelation occurs.

Polyoxyalkylene block copolymers are particularly preferred to use asthe biocompatible reverse-thermal gelation polymer. A polyoxyalkyleneblock copolymer is a polymer including at least one block (i.e. polymersegment) of a first polyoxyalkylene and at least one block of a secondpolyoxyalkylene, although other blocks may be present as well. POE-POPblock copolymers are one class of preferred polyoxyalkylene blockcopolymers for use as the biocompatible reverse-thermal gelation polymerin the delivery composition. POE-POP block copolymers include at leastone block of a polyoxyethylene and at least one block of apolyoxypropylene, although other blocks may be present as well. Thepolyoxyethylene block may be represented by the formula (C₂H₄O)_(b) whenb is an integer. The polyoxypropylene block may be represented by theformula (C₃H₆O)_(a) when a is an integer. The polyoxypropylene blockcould be for example (CH₂CH₂CH₂O)_(a), or could be

Several POE-POP block copolymers are known to exhibit reverse-thermalgelation properties, and these polymers are particularly preferred forimparting reverse-thermal viscosity behavior properties to the deliverycomposition of the present invention. Examples of POE-POP blockcopolymers include Pluronic™ F68, Pluronic™ F127, Pluronic™ L121,Pluronic™ L100, and Tetronic™ T1501. Tetronic™ T1501 is one example of aPOE-POP block copolymer having at least one polymer segment in additionto the polyoxyethylene and polyoxypropylene segments. Tetronic™ T1501 isreported by BASF Corporation to be a block copolymer including polymersegments, or blocks, of ethylene oxide, propylene oxide and ethylenediamine.

As will be appreciated, any number of biocompatible polymers may now orhereafter exist that are capable of imparting the desiredreverse-thermal viscosity behavior to the delivery composition of thepresent invention, and such polymers are specifically intended to bewithin the scope of the present invention when incorporated into thedelivery composition.

Some preferred POE-POP block copolymers have the formula:

HO(C₂H₄₀)_(b)(C₃H₆₀)_(a)(C₂H₄₀)_(b)H  I

which, in the preferred embodiment, has the property of being liquid atambient or lower temperatures and existing as a semi-solid gel atmammalian body temperatures wherein a and b are integers in the range of15 to 80 and 50 to 150, respectively. A particularly preferred POE-POPblock copolymer for use with the present invention has the followingformula:

HO(CH₂CH₂O)_(b)(CH₂(CH₃)CHO)_(a)(CH₂CH₂O)_(b)H  II

wherein a and b are integers such that the hydrophobe base representedby (CH₂(CH₃)CHO)_(a) has a molecular weight of about 4,000, asdetermined by hydroxyl number; the polyoxyethylene chain constitutingabout 70 percent of the total number of monomeric units in the moleculeand where the copolymer has an average molecular weight of about 12,600atomic mass units (amu) or Daltons. Pluronic™ F-127, also known aspoloxamer 407, is such a material. In addition, a structurally similarPluronic™ F-68 may also be used.

The procedures used to prepare aqueous solutions which form gels ofpolyoxyalkylene block copolymer are well known and are disclosed in U.S.Pat. No. 5,861,174, which is incorporated herein by reference in itsentirety. Typically, the amount of polymer and the amount ofhematopoietic growth factor are selected such that the resultingcomposition has reverse-thermal gelation properties at a transitiontemperature at less than about 37° C., preferably between about 10° C.and 37° C., more preferably between about 20° C. to about 37° C. Theconcentration of the first biocompatible polymer in the composition willvary depending upon the specific first biocompatible polymer and thespecific situation. In most situations, however, the first biocompatiblepolymer will comprise from about 1% by weight to about 70% by weight,and more typically from about 10% by weight to about 33% by weight. Forexample, particularly preferred for Pluronic™ F-127 is a range of fromabout 13% by weight to about 25% by weight.

The second biocompatible polymer is a protective colloid, and isincluded to impart increased resistance of the delivery composition tophysical deterioration that might otherwise occur then the deliverycomposition encounters extraneous aqueous liquids. This is particularlyimportant to protect the higher-viscosity form, such as the gel, frompremature structural deterioration due to the influence of aqueousbiological fluids following administration to the host. In particular,the first biocompatible polymer is subject to dissolution in aqueousbiological liquids encountered after administration of the deliverycomposition to the host. The second biocompatible polymer helps toinhibit or prevent such dissolution of the first biocompatible polymer,thereby helping to maintain the structural integrity of the deliverycomposition in vivo.

The second biocompatible polymer is hydrophilic. Preferably, the secondbiocompatible polymer is more hydrophilic than the first biocompatiblepolymer. By having a higher affinity for water than the firstbiocompatible polymer, the second biocompatible polymer tends to protectthe first biocompatible polymer from being dissolved away by aqueousbiological fluids present in the host. The protection afforded by thesecond biocompatible polymer helps to inhibit deterioration of thedelivery composition, so that the higher-viscosity form of the deliverycomposition will endure for some significant time followingadministration, permitting delivery of the hematopoietic growth factorto be sustained over an extended time. Absent the second biocompatiblepolymer, the first biocompatible polymer would be much more susceptibleto dissolution by biological fluids, which could, for example,prematurely destroy integrity of the desired gel character of thehigh-viscosity form of the delivery composition.

Also, the second biocompatible polymer is of colloidal molecular sizeand of high molecular weight. Typically, the weight average molecularweight of the second biocompatible polymer is at least 5,000 Daltons andmore typically, at least 10,000 Daltons. In many situations, the secondbiocompatible polymer has a weight average molecular weight of at least50,000 and often 100,000 or more.

The second biocompatible polymer can be any biocompatible polymer thatacts as a protective colloid in the delivery composition. The secondbiocompatible polymer will, however, ordinarily be a saccharide-basedpolymer. By saccharide-based, it is meant that the second biocompatiblepolymer is a polysaccharide or a derivative of a polysaccharidematerial.

Cellulosic polymers are particularly preferred for use as the secondbiocompatible polymer, and especially preferred are cellulosic polymersthat are swellable by water. Nonlimiting examples of cellulosic polymersfor use as the second biocompatible polymer include methylcellulose,hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropyl methylcellulose, ethyl hydroxyethyl cellulose andcarboxymethyl cellulose. A particularly preferred cellulosic polymer foruse as the second biocompatible polymer is hydroxypropylmethylcellulose.

Also useful as the second biocompatible polymer are phycocolloids. Aphycocolloid is a hydrophilic carbohydrate polymer occurring in algae,and derivatives of such polymers. Some examples of phycocolloids includecarrageenan, algin, and agar. Also useful as the second biocompatiblepolymer are alginates such as, for example, sodium alginate.

The hematopoietic growth factor, liquid vehicle, first biocompatiblepolymer and second biocompatible polymer can be present in the deliverycomposition in any suitable relative proportions compatible with theperformance requirements of the delivery composition. Typically,however, the delivery composition will include from 0.00000001 weightpercent to 0.000005 weight percent of the hematopoietic growth factor,from 60 weight percent to 96 weight percent of the liquid vehicle, from5 weight percent to 33 weight percent of the first biocompatible polymerand from 0.1 weight percent to 5 weight percent of the secondbiocompatible polymer.

The composition of the present invention can also comprise otheradditives, including polymer or protein stabilizers such as glycerol,trehalose, sucrose, glycine, mannitol, and albumin.

In one embodiment, the composition may optionally also include an activeagent, in addition to the hematopoietic growth factor, that is to bedelivered to a host along with the hematopoietic growth factor. In onepreferred embodiment, the composition is used for vaccination purposes,and the composition includes an antigen in addition to the hematopoieticgrowth factor. As used herein, antigen refers to any substance ormaterial capable of causing an immune response when administered to ahost. Antigens include, for example, polypeptides, peptides, proteins,glycoproteins and polysaccharides that are obtained from animal, plant,bacterial, viral protozoan and parasitic sources or are produced bysynthetic methods, including epitopes of proteins.

Exemplary antigens which may be included in the composition of thepresent invention include antigens from bacteria, protozoa and virusesagainst which vaccines are currently available or later developed, suchas antigens from viruses, protozoa or bacteria that are the causativeagents of childhood illnesses, Tetanus toxoid, Diphtheria toxoid andother non-pathogenic mutants of these toxins, antigens from Bordatellapertussis, antigens from M. tuberculosis, antigens from P. falciparum,antigens from blood-borne pathogens including Hepatitis C antigens, andHIV antigens; tumor-specific antigens; and antigens derived from HCG orother molecules involved in the mammalian reproductive cycle. Preferablythe antigen is selected from the group consisting of tetanus toxoid,diphtheria toxoid and other non-pathogenic mutants of these toxins,other antigens from viruses or bacteria that are the causative agents ofchildhood illnesses, antigens from M. tuberculosis, antigens fromBordatella pertussis, antigens from viruses or bacteria against whichvaccines are currently available, Hepatitis C antigens, HIV antigens andantigens from other blood-borne pathogens and tumor-specific antigens.Most preferably the antigen is selected from the group consisting ofTetanus toxoid, Diphtheria toxoid and other non-pathogenic mutants ofthese toxins, other antigens from viruses or bacteria that are thecausative agents of childhood illnesses, antigens from M. tuberculosis,antigens from Bordatella pertussis or HIV and antigens from viruses orbacteria against which vaccines are currently available. Particularlypreferred is for the antigen to include one or more of tetanus toxoid,diphtheria toxoid and antigens from Bordatella pertussis.

When an antigen is used, the amount of antigen in the composition of thepresent invention varies depending on the nature and potency of theantigen. Typically, however, the amount of antigen present in thecomposition of the present invention is from about 0.000001% by weightof the composition to about 5% by weight of the composition.

The composition of the present invention may be administered to a hostto achieve any desired hematopoietic effect. Preferably the host is amammal, and more preferably a human. The composition can be administeredin a variety of forms adapted to the chosen route of administration,e.g., parenterally. Parenteral administration in this respect includesadministration by the following routes: intravenous, intramuscular,subcutaneous, intrasynovial, transepithelially including transdermal,sublingual and buccal, intranasal, and intraperitoneal. Preferably, themode of administering the composition of the present invention isselected from the group consisting of subcutaneous and intramuscularinjections, and mucosal routes, including intranasal, with injectionroutes being even more preferred.

The composition is typically prepared in water, a saline solution oranother aqueous liquid as the liquid vehicle. Under ordinary conditionsof storage and use, these preparations can also contain a preservativeto prevent the growth of microorganisms. The composition suitable forinjectable use includes sterile aqueous solutions. Preferably, thecomposition is sterile with sufficient fluidity for easy syringability.It can be stable under the conditions of manufacture and storage andpreferably preserved against the contaminating action of microorganismssuch as bacterial and fungi. The liquid vehicle can be a solvent ofdispersion medium containing, for example, water, ethanol, polyol (e.g.,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by maintaining the temperature of thecomposition having reverse-thermal gelation properties below thetransition temperature. The prevention of the action of microorganismscan be brought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, e.g., sugars, phosphate buffers, sodium chloride, or mixturesthereof.

The composition of the present invention can be implanted directly intothe body by injecting it as a liquid, whereupon the composition willordinarily gel as it warms inside the body when the composition hasreverse-thermal gelation properties. Also, although the composition ofthe present invention is typically sufficiently fluid and flowable whenadministered, it will convert to a semi-solid gel inside the host whenit has the proper reverse-thermal gelation properties. Also, thecomposition can be administered in the form of a gel, for example bysurgical implantation of the gel.

In another embodiment, solutes, can be incorporated into the deliverycomposition of the present invention, for example to stabilize thehematopoietic growth factor. The use of such protein-stabilizingsolutes, such as, for example, sucrose, not only aid in protecting andstabilizing the protein (i.e., hematopoietic growth factor), but alsoallow the first biocompatible polymer to form suitable gels at lowerconcentrations than needed in water or buffer alone and/or to change thetransition temperature at which reverse-thermal gelation occurs. Thus,the working range of first biocompatible polymer concentration can bewidened and the transition temperature modified. It is known that insome cases a gel will not form when the concentration ofpolyoxyethylene-polyoxypropylene block copolymer in water or dilutebuffer is outside a particular range, e.g., equal to or less than 15%percent by weight for some such polymers. However, by introducingprotein-stabilizing solutes into the composition of the presentinvention, the transition temperature may be manipulated, while alsolowering the concentration of polyoxyethylene-polyoxypropylene blockcopolymer that is necessary to form a gel.

The hematopoietic growth factor delivery composition can be used tostimulate hematopoietic cell activity by administering the growth factordelivery composition to a host by any suitable administration technique.Typically, the delivery composition is chilled at the time ofadministration so as to be in the form of a flowable medium. Also, thefirst biocompatible polymer, and the hematopoietic growth factor, ispreferably substantially entirely dissolved in the liquid vehicle whenadministered to the host.

In another aspect of the invention, a method is provided for packagingand storing the hematopoietic growth factor delivery composition.According to this aspect of the invention, the delivery composition isplaced in a container when the composition is in the form of a flowablemedium. The temperature of the composition is then raised so that thedelivery composition converts to a gel form within the container forstorage. Following storage in the gel form, the delivery composition canbe converted back to a flowable medium for administration to the host atthe appropriate time by lowering the temperature of the deliverycomposition in the container. In this way, the delivery composition iseasy to handle during manufacturing and packaging operations, but can bestored in the highly stable form of a gel. Furthermore, the deliverycomposition can be converted back to a flowable medium for ease ofadministration. This ability to store the hematopoietic growth factor inthe protective gel form prior to use is a distinct advantage with thepresent invention. Alternatively, the delivery composition could bestored in the form of a flowable medium at a temperature below thereverse-thermal liquid-gel transition temperature, but such a flowablemedium is often not as convenient for handling and storage as a gelform.

In another aspect, a method for making the delivery composition isprovided, comprising dissolving the first biocompatible polymer in aliquid vehicle and suspending or codissolving the hematopoietic growthfactor and the second biocompatible polymer in the liquid vehicle.Preferably, both the hematopoietic growth factor and the secondbiocompatible polymer are also dissolved in the liquid vehicle duringthe manufacture.

EXAMPLES

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which is not intended to be limiting.

Example 1 Formulation of G-CSF with Pluronic™ F127

In one preferred embodiment of the present invention, the hematopoieticgrowth factor is G-CSF, and the delivery composition of the presentinvention provides a delivery system for the sustained administration ofG-CSF to a human or animal host. A preferred first biocompatible polymerin this situation is a POE-POP block copolymer with reverse-thermalgelation properties.

As a specific formulation example, G-CSF can be formulated withPluronic™ F127 (poloxamer 407), with and withouthydroxypropylmethylcellulose (HPMC). Dry powder forms of Pluronic™ F127and HPMC are weighed, mixed together, and then reconstituted in water orphysiological buffer to achieve the drug delivery matrix containing,upon addition of G-CSF, the desired concentrations of each component.More specifically, the concentration of Pluronic™ F127 is one that willachieve a final concentration (e.g., 5-30 weight %) at which it forms asemi-solid gel, along with the addition of BPMC, at body temperature(37° C.). If included, HPMC can be added in an amount to achieve a finalconcentration (e.g., HPMC 0.1-5 weight %) necessary to modulate thephysicochemical or pharmacological properties of the Pluronic™ F127 orG-CSF. Alternatively, Pluronic™ F127 and HPMC can be formulatedseparately as individual solutions and then mixed together to producethe drug delivery matrix containing, upon addition of G-CSF, the desiredconcentrations of each component. As a further alternative, a solutionof either Pluronic™ F127 or HPMC in buffer or water and a dry powder ofthe second polymer (i.e., either Pluronic™ F127 or HPMC) can be mixedtogether to achieve the drug delivery matrix containing, upon additionof G-CSF, the desired concentrations of each component.

G-CSF can be added to the liquid or dry mixture of Pluronic™ F127 andHPMC. The G-CSF can be added in dry powder form, or as a liquid solutionto the drug delivery matrix. Final concentrations of G-CSF in thePluronic™ F127 and HPMC drug delivery matrix include thoseconcentrations that will provide biological levels of G-CSF as asustained release following injection. For example, G-CSF can be addedat a concentration so that the injected volume contains dosages of G-CSFthat would provide therapeutic levels for a sustained period afteradministration. More specifically, G-CSF can be incorporated into thedelivery matrix at various desired concentrations, such as for example,to provide 1 to 500 μg/injection of G-CSF.

The addition of HPMC in the delivery matrix modulates G-CSF in such away that the pharmacokinetic profile is altered to provide sustainedlevels of G-CSF in serum compared to T G-CSF in only Pluronic™ F127.Furthermore, addition of HPMC greatly increases the pharmacologicalaction of G-CSF not only on peripheral blood hematopoeisis, but also onspleen and bone marrow cell hematopoeisis. Although the mechanism ofaction of HPMC on the pharmacokinetic profile and pharmacological actionof G-CSF is not known, it is proposed that the addition of HPMC may 1)stabilize the delivery matrix, 2) stabilize the hematopoietic growthfactor, 3) target the hematopoietic growth factor to its site of actionwithin the body, and/or 4) enable the hematopoeitic growth factor toexert its hematopoeitic action on earlier progenitor cells eitherdirectly by the growth factor or indirectly by stimulating othercytokines or growth factors.

Example 2 Administration of G-CSF with Pluronic™ 127

Formulations including G-CSF, Pluronic™ 127, with and without HPMC, areprepared and administered to groups of Balb/c mice to determine a) theeffect of formulating G-CSF in a Pluronic™ 127 and HPMC (InventionFormulation) delivery matrix on the pharmacokinetic profile of G-CSFcompared to conventionally (Buffer Formulation) formulated G-CSF and b)the effects of the Invention Formulation on hematopoietic activitycompared to conventionally formulated G-CSF. The formulations areadministered to mice intramuscularly (i.m.), as a single dose forpharmacokinetic analysis and as either single (for InventionFormulation) or multiple (for Buffer Formulation) doses forhematopoeitic activity. The compositions of the formulations are shownin Table 1.

TABLE 1 Pluronic ™ F127 (% G-CSF HPMC (% Group w/w) (μg/mL) w/w) Vehiclecontrol, 0 0 0 buffer Vehicle control, gel 17 0 0.1 to 5 G-CSF in buffer0 1 to 300 0 (Buffer Formulation) G-CSF with 17 7 to 100 0.1 to 5Pluronic ™ 127 and HPMC (Invention Formulation)

Two studies are performed. In the first study, Balb/c mice receive asingle i.m. injection of 7 μg G-CSF in either the Buffer Formulation orthe Invention Formulation. Blood is sampled at various timepoints for upto 96 h. G-CSF concentration in serum is determined via ELISA. Thepharmacokinetic profile of G-CSF from the Buffer Formulation and theInvention Formulation are shown in FIG. 1. In the second study, a groupof mice receive a single i.m. injection of G-CSF in the InventionFormulation at a dose of 0.7, 3.5 or 35 μg/mouse, and another group ofmice receive 7 injections of G-CSF in the Buffer Formulation at a doseof 0.1, 0.5 or 5 μg/injection for a total cumulative dose of 0.7, 3.5 or35 μg/mouse. The dosing schedule and study design for this second studyis outlined in FIG. 2. The hematopoietic action of these two G-CSFformulations as well as vehicle controls was determined by assessingnumbers of HPP-CFC cells in the peripheral blood leukocyte fraction 4days after initiation of G-CSF injection. HPP-CFC cells were quantitatedmicroscopically after performing colony forming assays of cytokine(IL-3)-stimulated peripheral blood leukocytes. The results of thissecond study are shown in FIG. 3.

To summarize the results in the two studies, G-CSF in the InventionFormulation:

-   -   (a) has a longer serum half-life; and    -   (b) increases HPP-CFC numbers in peripheral blood better than        conventionally formulated and administered G-CSF.

The results show a substantial improvement in the delivery of G-CSF,using the Invention Formulation relative to the Buffer Formulation.Referring to FIG. 3, it is particularly surprising and noteworthy thatthe Invention Formulation administered in a single 3.5 μg of G-CSFinjection increased the presence of HPP-CFC in peripheral blood by anamount comparable to the increase produced by 10 times as much G-CSF (35μg) administered in a multi-injection regimen using the BufferFormulation. The improvement in delivery of G-CSF can be seen in atleast two specific ways: first by providing a more sustainedpharmacokinetic profile (longer half-life) and second as creating anapparently more potent hematopoietic growth factor than is exhibited byconventional formulations. The importance of this can been viewed from avariety of perspectives, including:

-   -   (a) as a component to a marketed hematopoeitic growth factor, it        would likely decrease dose costs of since less growth factor        would likely be used to produce the same biological effect;    -   (b) from the patient perspective, if a total lower drug dosage        is used, it would be expected to produce fewer side effects        (which for hematopoeitic growth factors are often dose limiting,        including fever and joint pain); and    -   (c) also from the patient perspective, a better pharmacokinetic        profile leads to fewer injections per course of therapy.

Example 3 Formulation of Flt3-L with Pluronic™ F127

In a preferred embodiment of the present invention, the hematopoieticgrowth factor is Flt3-L, and the pharmaceutical composition of thepresent invention provides a delivery system for the sustainedadministration of the Flt3-L to a human or animal. A preferred firstbiocompatible polymer in this situation is a POE-POP block copolymerwith reverse-thermal gelation properties.

As a specific formulation example, Flt3-L can be formulated withPluronic™ F127 (poloxamer 407), with or withouthydroxypropylmethylcellulose (HPMC). Pluronic™ F127 is initiallyformulated in water or physiological buffer at concentrations (e.g.,5-30%) at which it forms a semi-solid gel, along with the addition ofHPMC, at body temperature (37° C.). HPMC may then be added to thePluronic™ F127 formulation at concentrations necessary to modulate thephysicochemical properties of the Pluronic™ F127. (e.g., finalconcentrations of HPMC₁₋₅%). Alternatively, Pluronic™ F127 and BPMC canbe formulated separately as individual solutions and then mixed togetherto produce the drug delivery matrix containing, upon addition of Flt3-L,the desired concentrations of each component. As a further alternative,dry powder forms of Pluronic™ F127 and HPMC can be mixed together andthen reconstituted in water or physiological buffer to achieve the drugdelivery matrix containing, upon addition of Flt3-L, the desiredconcentrations of each component.

Flt3-L can be added to the liquid or dry mixture of Pluronic™ F127 andHPMC. The Flt3-L can be added in dry powder form, or as a liquidsolution to the drug delivery matrix. Final concentrations of Flt3-L inthe Pluronic™ F127 and HPMC drug delivery matrix include thoseconcentrations that will provide biological levels of Flt3-L as asustained release following injection. For example, Flt3-L could beadded at concentrations ranging from about 3 to about 15 μg for proposeddelivery of 1 to 5 μg per day over a 3 day sustained release.

The addition of HPMC modulates Flt3-L in this delivery formulation insuch a way that although the pharmacokinetic profile of Flt3-L in serumis not altered compared to a drug delivery matrix containing onlyPluronic™ F127, the biological activity of Flt3-L on spleen and bonemarrow cell differentiation is significantly increased.

Example 4 Administration of Flt3-L with Pluronic™ 127

Formulations including Flt3-L, Pluronic™ 127 and, optionally, HPMC areadministered to groups of Balb/c mice to determine a) the effects of theformulations on pharmacokinetics compared to conventionally formulatedFlt3-L and b) the effects of the formulations on hematopoietic activitycompared to conventionally formulated Flt3-L. The formulations areadministered to mice i.m. as a single dose. The compositions of theformulations are shown in Table 2.

TABLE 2 Pluronic ™ Group F127 % w/w Flt3-L (ug/mL) HPMC % (w/w) Vehiclecontrol 0 0 0 (aqueous buffer) Flt-3L parent 0 150 0 (Flt3-L formulatedin aqueous buffer) PGZ-1 22 150 0 (Flt3-L formulated in F127) PGZ-2 22150 5 (Flt3-L formulated in F127/HPMC)

In a study, mice are sacrificed daily up to 4 days after injection andplasma is collected to determine drug pharmacokinetics as well ascirculating and splenic white blood cell counts. The pharmacokineticprofile of Flt3-L is shown in FIG. 4. FIGS. 5A and 5B show circulatingwhite blood cells (WBC) and spleen cell counts (SPC). Particularlysignificant are the much higher white blood cell and spleen cell countsrecorded in the case of PGZ-1 vs. PGZ-2, as shown in FIGS. 5A and 5B.This is particularly noteworthy because both formulations exhibitsimilar pharmacokinetic profiles, as shown in FIG. 4.

To summarize the results in the above-described studies, formulationsmade according to the present invention:

-   a) have a longer plasma half-life;-   b) increase white cells to a greater extent at equivalent doses    compared to conventionally formulated and administered Flt3-L; and-   c) increase CFU-GM and HPP-CFC in both the bone marrow and spleen    better than conventionally formulated and administered Flt3-L.

The results show a substantial improvement in the delivery of theFlt3-L, using the invention including HPMC being exceptionally good. Theimprovement can be seen in at least two specific ways, first byproviding a more sustained pharmacokinetic profile (longer half-life)and secondly as creating an apparently more potent hematopoietic growthfactor than is exhibited by the other formulations. The importance ofthis can been viewed from a variety of perspectives, including:

-   a) as a component to a marketed hematopoeitic growth factor, it    would likely decrease dose costs of since less growth factor would    likely be used to produce the same biological effect;-   b) from the patient perspective, if a total lower drug dosage is    used, it would be expected to produce fewer side effects (which for    hematopoeitic growth factors are often dose limiting, including    fever and joint pain); and-   C) also from the patient perspective, a better pharmacokinetic    profile leads to fewer injections per course of therapy.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

1. A hematopoietic growth factor delivery composition, the compositioncomprising: a hematopoietic growth factor capable of stimulatinghematopoietic cell activity when administered to a mammalian host; afirst biocompatible polymer and a liquid vehicle in which the firstbiocompatible polymer is at least partially soluble at some temperature,the first biocompatible polymer interacting with the liquid vehicle toimpart reverse thermal viscosity behavior to the composition over atleast some temperature range, so that the composition is in alower-viscosity form when the temperature of the composition is at afirst temperature within the range and the composition is in ahigher-viscosity form when the temperature is at second temperaturewithin the range that is higher than the first temperature; and a secondbiocompatible polymer being a protective colloid that inhibits thedissolution into aqueous liquids of the first biocompatible polymer atleast when the composition is in the higher-viscosity form; wherein theliquid vehicle comprises from 60 weight percent to 96 weight percent ofthe composition, the first biocompatible polymer comprises from 5 weightpercent to 33 weight percent of the composition, and the secondbiocompatible polymer comprises from 0.1 weight percent to 5 weightpercent of the composition; and wherein the hematopoietic growth factorcomprises a member selected from the group consisting, of granulocytemacrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) andFlt3 ligand (Flt3-L); and wherein, the first biocompatible polymercomprises a polyoxyalkylene block copolymer comprising at least oneblock of a polyoxyethylene and at least one block of a polyoxypropylene,and the second biocompatible polymer comprises a cellulosic polymer. 2.The hematopoietic growth factor delivery composition of claim 1, whereinthe first temperature is lower than 20° C. and the second temperature ishigher than 25° C.
 3. The hematopoietic growth factor deliverycomposition of claim 2, wherein the first temperature is in a range offrom 1° C. to 20° C. and the second temperature is higher than 25° C. 4.The hematopoietic growth factor delivery composition of claim 3 whereinthe second temperature is 37° C.
 5. The hematopoietic growth factordelivery composition of claim 4, wherein the higher-viscosity form has aviscosity that is at least 3 times as large as the viscosity of thelower-viscosity form.
 6. The hematopoietic growth factor deliverycomposition of claim 1, wherein the lower-viscosity form is a flowablemedium and the higher-viscosity form is a gel.
 7. The hematopoieticgrowth factor delivery composition of claim 6, wherein the cellulosicpolymer has an affinity for water such that the cellulosic polymerinhibits deterioration of the gel by invasion of the composition byaqueous biologic fluids when the composition is administered to abiologic host. 8-13. (canceled)
 14. The hematopoietic growth factordelivery composition of claim 1, wherein the polyoxyethylene comprisesat least 70 weight percent of the first biocompatible polymer.
 15. Thehematopoietic growth factor delivery composition of claim 1, wherein thepolyoxypropylene has the formula (C₃H₆O)_(b), where b is an integer. 16.The hematopoietic growth factor delivery composition of claim 1, whereinthe polyoxypropylene has the formula

where b is an integer.
 17. The hematopoietic growth factor deliverycomposition of claim 1, wherein the cellulosic polymer has a weightaverage molecular weight of at least 5,000 Daltons. 18-19. (canceled)20. The hematopoietic growth factor delivery composition of claim 1,wherein the cellulosic polymer comprises methylcellulose.
 21. Thehematopoietic growth factor delivery composition of claim 1, wherein thecellulosic polymer comprises hydroxymethylcellulose.
 22. Thehematopoietic growth factor delivery composition of claim 1, wherein thecellulosic polymer comprises hydroxyethylcellulose.
 23. Thehematopoietic growth factor delivery composition of claim 1, wherein thecellulosic polymer comprises hydroxypropyl cellulose.
 24. Thehematopoietic growth factor delivery composition of claim 1, wherein thecellulosic polymer comprises hydroxypropyl methylcellulose.
 25. Thehematopoietic growth factor delivery composition of claim 1, wherein thecellulosic polymer comprises carboxymethylcellulose.
 26. Thehematopoietic growth factor delivery composition of claim 1, wherein thecellulosic polymer comprises ethyl hydroxyethyl cellulose. 27-32.(canceled)
 33. The hematopoietic growth factor delivery composition ofclaim 1, wherein the cellulosic polymer has a weight average molecularweight of at least 10,000 Daltons.
 34. The hematopoietic growth factordelivery of claim 1, wherein the liquid vehicle is an aqueous liquid.35-46. (canceled)
 47. The hematopoietic growth factor deliverycomposition of claim 1, comprising an antigen that is different than thehematopoietic growth factor.
 48. (canceled)
 49. The hematopoietic growthfactor delivery composition of claim 1, wherein the composition iscontained within an injection device that is actuatable to administerthe composition to the host by injection.
 50. A method of packaging andstoring the hematopoietic growth factor delivery composition of claim 6,comprising placing the composition in a container when the compositionis in the form of the flowable medium and, after the placing, raisingthe temperature of the composition in the container to convert thecomposition to the gel for storage, wherein the gel form in thecontainer can be converted back to the form of a flowable medium foradministration to the host by lowering the temperature of thecomposition in the container.
 51. (canceled)
 52. The hematopoieticgrowth factor delivery composition of claim 1, wherein the host is ahuman.
 53. The hematopoietic growth factor delivery composition of claim1, wherein the first biocompatible polymer is poloxamer 407.